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Biomedical subjects

L Inverardi

Publications and source records attributed to L Inverardi.

At least 55 records · Page 3Linked to original sources
PubMed1997

Improved human islet isolation using a new enzyme blend, liberase.

Enzymatic digestion of donor pancreases is a vital step in human and large mammalian islet isolation. The variable enzymatic activities of different batches of commercially available collagenase is a major obstacle in achieving reproducibility in islet isolation procedures. In the present work, the effectiveness of Liberase, a standardized mixture of highly purified enzymes recently developed for the separation of human islets, was compared with that of a traditional collagenase preparation (type P). The results of 50 islet isolations using Liberase enzyme were compared with those of 36 isolations with collagenase, type P. No significant differences in donor age, cold ischemia time, digestion time, or weight of the pancreases were observed between the two groups. Islet yield was significantly higher in the group where the Liberase enzyme was used. All parameters examined (islet number, islet number per gram of tissue, islet equivalent number, and islet equivalent number per gram of tissue) were significantly improved when Liberase enzyme was used. Different lots of Liberase enzyme were tested, and no difference was observed. Islets isolated with Liberase enzyme were also of larger size and were much less fragmented, suggesting a gentler enzymatic action and better preservation of anatomical integrity. Islets isolated with Liberase enzyme, assessed both in vitro and in vivo, revealed a functional profile similar to that of islets separated with collagenase. Liberase enzyme appears, therefore, to represent a new powerful tool for improving the quality of human islet isolation.

Adult↗
PubMed1996

The role of the perforin and Fas pathways of cytotoxicity in skin graft rejection.

Cell-mediated cytotoxicity is believed to play an important role in the mechanisms of rejection following organ and tissue transplantation. We have investigated the role of the perforin and Fas pathways of cell-mediated cytotoxicity on the survival of xenogeneic, allogeneic and hemiallogeneic skin grafts. Wild type C57BL/6 (B6), perforin deficient C57BL/6 (B6 PKO) and Fas ligand deficient (B6 gld/gld) mice were grafted with xenogeneic Lewis rat skin grafts, or allogeneic B10.BR, class I disparate B6 bml, class II disparate B6 bm12, and Fas deficient allogeneic C3H lpr/lpr grafts. No significant difference was observed in the timing of rejection with B6 PKO mice as recipients when compared with normal B6 controls. All grafts were also rejected by B6 gld/gld recipients, but with a significantly delayed pattern of rejection, showing, therefore, that Fas, although not critical, may participate to the mechanisms of rejection. Neither perforin nor Fas, however, seem to be essential for rejection of a skin graft, and it is likely that alternative mechanisms assume an important role in the effector phase of rejection once these cell-cytotoxic pathways are abrogated.

Animals↗
PubMed1996

Feedback modulation of ligand-engaged alpha L/beta 2 leukocyte integrin (LFA-1) by cyclic AMP-dependent protein kinase.

Rapid interconversion between a firmly adherent and a nonadherent, circulating phenotype is a distinctive feature of mature leukocytes and is thought to be essential for efficient immune surveillance. Leukocyte adhesion is a finely regulated process controlled in part by reversible, activation-dependent up-regulation of beta 1- and beta 2- integrin function. To investigate the molecular basis of such reversibility in human T lymphocytes, we developed a model of alpha L/beta 2 (LFA-1)-dependent adhesion that uses a heterologous cell line expressing human intercellular adhesion molecule-1 as a selected ligand. We show here that intracellular cAMP elevation, followed by cAMP-dependent kinase activation, promotes T cell deadhesion by disassembling the actin-based cytoskeleton, thus dissociating LFA-1 from cytoskeletal anchoring proteins that normally connect the adhesion receptor to F-actin in lymphocytes engaged in intercellular adhesion. Cells costimulated via the CD3 and LFA-1 receptors by specific Abs or by binding to intercellular adhesion molecule-1 display gradual and persistent intracellular cAMP elevations due to the synergistic induction of a protein kinase C-dependent adenylyl cyclase isoform. On the basis of these findings, we propose a feedback model for short term regulation of leukocyte integrins, involving sequential, integrin-dependent activation of the protein kinase C and adenylyl cyclase/cAMP-dependent kinase enzymatic pathways and leading to disengagement of the adhesion receptor from its specific ligand.

Actins↗
PubMed1995

Conserved regions in the cytoplasmic domains of the leukocyte integrin alpha L beta 2 are involved in endoplasmic reticulum retention, dimerization, and cytoskeletal association.

Selected functions of integrins, including regulated cytoskeletal association and transmembrane signaling, depend on a poorly defined bidirectional communication between the extracellular and cytoplasmic domains of the alpha and beta subunits. To investigate this problem in the leukocyte integrin alpha L beta 2 (LFA-1), we generated a series of cytoplasmic truncation or internal substitution mutants of the alpha L and beta 2 cytoplasmic domains, and assessed their biochemical and functional properties upon ectopic expression in constitutively adherent cells. Expression of the alpha L beta 2 heterodimer in stably adherent cells is sufficient to promote its constitutive cytoskeletal association. Structural determinants for such association are located in selected regions of the beta 2 cytoplasmic domain, which display functional interdependence. In addition, a conserved region (Arg733-Lys742) in the beta 2 cytoplasmic domain seems to be critical not only for its cytoskeletal association, but also for endoplasmic reticulum retention, assembly, and transport to the plasma membrane of the mature alpha L beta 2 heterodimer. Analysis of deletion mutants of the alpha L subunit demonstrates a role of the conserved, membrane-proximal GFFKR motif in conferring stability to the alpha beta complex, possibly because of its direct involvement in heterodimer formation. We propose that a previously uncharacterized association of defined subregions of the cytoplasmic domains of integrin alpha and beta subunits affects the dimerization and regulated function of the adhesion receptor.

Allosteric Regulation↗
PubMed1994

Early events in cell-mediated recognition of vascularized xenografts: cooperative interactions between selected lymphocyte subsets and natural antibodies.

Cell-mediated early immune recognition of xenogeneic vascularized discordant grafts is poorly characterized. It has been the purpose of our studies to elucidate the role of lymphocytes in the recognition and rejection phenomena. To this end, we have utilized both ex vivo and in vitro model systems. We demonstrate that selected human lymphocyte subpopulations (mainly NK cells) rapidly and specifically adhere to xenogeneic endothelia. Efficient lysis of endothelial cells is subsequently mediated by such a population. Adhesion and cytotoxicity occur via at least two pathways, one dependent on and the other independent of the presence of human natural antibodies of the G class. Both IgG-dependent and IgG-independent adhesion and cytotoxicity can be partly inhibited by the use of anti-leukocyte integrin monoclonal antibodies. IgG-dependent adhesion and cytotoxicity can be also partly inhibited by carbohydrate structures that contain the alpha-galactosyl epitope. A possible role of these events in the eventual outcome of discordant vascularized xenogeneic transplants can be postulated.

Animals↗
PubMed1992

Early recognition of a discordant xenogeneic organ by human circulating lymphocytes.

Cell-mediated immune mechanisms underlying discordant xenograft rejection are poorly characterized. In our study, using a human to rat xenogeneic ex vivo model, we show that a fraction of human lymphocytes, when perfused through the coronary system of a rat heart, rapidly and specifically adheres to the vascular endothelium and infiltrates the myocardium. Lymphocyte phenotypic analysis before and after perfusion, as well as the use of purified cell subpopulations, demonstrate preferential adhesion of CD3- CD16+ NK cells. NK cell adhesion occurs via xenoreactive antibody-dependent and -independent pathways, because the selective removal of human IgG from the perfusion buffer markedly reduces but does not completely abrogate NK cell sequestration. However, T lymphocytes are retained in the xenoorgan via an antibody-independent pathway, as assessed by the lack of influence of IgG removal. Leukocyte integrins appear to play a crucial role in mediating adhesion of both lymphocyte subsets, because the pretreatment of lymphocytes with anti-CD11a, anti-CD11b, and anti-CD18 antibodies markedly reduces their retention into the xenogeneic organ. Retained human lymphocytes mediate rapid and direct damage of the xenoorgan, as demonstrated by histologic and functional alterations of the endothelium, impaired vascular resistance and in vitro lysis of rat endothelial cells by human NK cells. Taken together, these findings suggest a role for cell-mediated mechanisms in the rapid recognition and rejection of vascularized xenografts.

Animals↗